DESCRIPTION: To maximize clinical information content obtainable from a single small patient sample (e.g. cerebrospinal fluid-CSF), single molecule detection (SMD) technologies are the most promising for development of next- generation medical diagnostics. Recently, microRNAs (miRNAs) have been implicated in human malignancies including neurodegenerative disorders, offering the exciting prospect to combine traditional protein markers with novel miRNA candidate markers in panels. But as patient samples are limited and marker concentrations often very low, serial analysis is not practical. Thus, only very sensitive methodologies with high multiplexing power will be able to maximize diagnostic value and be suitable for improved tests for early disease detection. Nesher Technologies, Inc. (NTI) has exclusively licensed the intellectual property for a revolutionary, quantitative, ultrasensitive and -specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with exquisite single-well multiplexing potential, minimal sample requirements, and extremely simplified workflows (no separation/washing and amplification steps). It is based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, whereby target recognition molecules are tagged with different color fluorescent dyes (and quenchers), allowing, on the same platform, ultrasensitive detection of both proteins and nucleic acids (including miRNAs) in body fluids. NTI recently achieved extension from 2-color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power (particularly when involving FRET), and demonstrated diagnostic utility for ultrasensitive protein as well as miRNA quantification at clinically relevant concentrations without amplification. Furthermore, recent work by our consultants Profs. Steve Quake and Shimon Weiss shows i) combination of microfluidics- based sample handling with ALEX spectroscopy, a new breakthrough approach for assay miniaturization termed single molecule opt fluidics, and ii) enhanced throughput using a multifocal excitation/detection geometry. NTI's long-term goal is to develop rapid, highly multiplexed (with a capacity of >100 analytes per sample), ultrasensitive and -specific, quantitative, cost-effective, and fully automated, protein- and nucleic acid- based diagnostic tests that require minimal sample sizes. Here we propose assay development and clinical validation for improved early-stage Alzheimer's disease (AD) diagnosis, implementing a panel of candidate protein and miRNA biomarkers. Our Specific Aims are: 1. Reagent development for a multiplex protein & microRNA biomarker-based next-generation AD test 2. Separate as well as multiplexed biomarker detection and quantification using spiked samples 3. ALEX-based analysis of archived clinical samples from 108 patients (PRoBE study design implementation) SBIR Phase II will propose assay expansion, miniaturization, and development of a versatile, user-friendly, diagnostic system as useful tool for early detection of AD and other neurodegenerative disorders.